The long-term objective of the proposed research is to better understand how enzymes activate stable covalent bonds. A mechanistic investigation of C-H bond activation in alternative dihydrofolate reductases (DHFRs) and thymidylate synthase (TSase) will be conducted. These enzymes play crucial roles in DNA biosynthesis and thus serve as targets for antibiotic and chemotherapeutic drugs. They are also model systems used to address fundamental issues in enzymology, such as the role of protein dynamics and environmentally coupled quantum mechanical hydrogen tunneling in bond activation. Aim 1: The FolA encoded chromosomal DHFR (cDHFR) is a small protein that catalyzes a single chemical transformation and has been studied extensively during the previous grant period. Methods were developed to study the physical nature of the C-H-C transfer. It has been demonstrated that the cDHFR reaction coordinate is perfectly arranged for H-tunneling, and that mutants far from the active site can synergistically disturb the H-transfer process. These findings suggested a network of coupled motions across the enzyme that enhance the catalyzed reaction. The proposed studies will extend these studies to compare the effects of different protein scaffolds, dynamics, and reactants orientation on the physical nature of the C-H-C transfer. Aim 2: TSase is larger than cDHFR and catalyzes the making and breaking of multiple covalent bonds. We will examine different C-H activation steps in the complex TSase catalyzed reaction. These studies will include the examination of kinetic and dynamic effects of controlled active site mutations on different H- transfer steps. We will also examine the effect of altered networks of coupled motions on kinetics and dynamics using mutations distal to the active site. The findings from kinetic methods that can expose the nature of specific H-transfer steps will be correlated to measurements of protein dynamics, to assess the role of motions in enhancing C-H bond activation, in both fast and rate-limiting chemical steps.